Identification of a novel LFNG variant in a Chinese fetus with spondylocostal dysostosis and a systematic review.

Spondylocostal dysostosis (SCDO) encompasses a group of skeletal disorders characterized by multiple segmentation defects in the vertebrae and ribs. SCDO has a complex genetic etiology. This study aimed to analyze and identify pathogenic variants in a fetus with SCDO. Copy number variant sequencing and whole exome sequencing were performed on a Chinese fetus with SCDO, followed by bioinformatics analyses, in vitro functional assays and a systematic review on the reported SCDO cases with LFNG pathogenic variants. Ultrasound examinations in utero exhibited that the fetus had vertebral malformation, scoliosis and tethered cord, but rib malformation was not evident. We found a novel homozygous variant (c.1078 C > T, p.R360C) within the last exon of LFNG. The variant was predicted to cause loss of function of LFNG by in silico prediction tools, which was confirmed by an in vitro assay of LFNG enzyme activity. The systematic review listed a total of 20 variants of LFNG in SCDO. The mutational spectrum spans across all exons of LFNG except the last one. This study reported the first Chinese case of LFNG-related SCDO, revealing the prenatal phenotypes and expanding the mutational spectrum of the disorder.

USP40 promotes hepatocellular carcinoma progression through a YAP/USP40 positive feedback loop.

Yes-associated protein (YAP) is an essential driver of hepatocellular carcinoma (HCC) progression and the ubiquitin-proteasome system controls its abundance. However, the role of ubiquitin-specific protease 40 (USP40) in YAP stability remains unclear. Here, USP40 was first identified as a novel regulator of YAP abundance and its target genes in HCC cells. USP40 interacted with YAP to remove the lysine 48 (K48)-linked polyubiquitination of YAP at K252 and K315 sites, thereby maintaining YAP stability. USP40 facilitated the proliferation, colony formation, migration and spheroid formation of HCC cells in vitro and promoted HCC growth in vivo in a YAP-dependent manner. In turn, YAP transcriptionally activated USP40 expression in HCC cells. RNA sequencing analysis showed that about 37% of USP40-regulated genes overlapped with YAP-regulated genes. Interestingly, stiffness-induced USP40 upregulation was abolished by YAP knockdown, and USP40 knockdown attenuated stiffness-induced YAP accumulation in HCC cells. Clinical data demonstrated that USP40 was positively associated with YAP expression in HCC tissues and its high expression indicated a poor prognosis. In conclusion, the USP40/YAP positive feedback loop contributes to HCC progression, suggesting that USP40 may be a promising drug target for anti-HCC.

CK2-mediated phosphorylation of SUZ12 promotes PRC2 function by stabilizing enzyme active site.

Polycomb repressive complex 2 (PRC2) plays a key role in maintaining cell identity during differentiation. Methyltransferase activity of PRC2 on histone H3 lysine 27 is regulated by diverse cellular mechanisms, including posttranslational modification. Here, we report a unique phosphorylation-dependent mechanism stimulating PRC2 enzymatic activity. Residue S583 of SUZ12 is phosphorylated by casein kinase 2 (CK2) in cells. A crystal structure captures phosphorylation in action: the flexible phosphorylation-dependent stimulation loop harboring S583 becomes engaged with the catalytic SET domain through a phosphoserine-centered interaction network, stabilizing the enzyme active site and in particular S-adenosyl-methionine (SAM)-binding pocket. CK2-mediated S583 phosphorylation promotes catalysis by enhancing PRC2 binding to SAM and nucleosomal substrates and facilitates reporter gene repression. Loss of S583 phosphorylation impedes PRC2 recruitment and H3K27me3 deposition in pluripotent mESCs and compromises the ability of PRC2 to maintain differentiated cell identity.

Clinical characteristics and variant analyses of transient infantile hypertriglyceridemia related to GPD1 gene.

Objective : Our study aims to summarize and analyze the clinical characteristics of transient infantile hypertriglyceridemia (HTGTI) and variants in the glycerol-3-phosphate dehydrogenase 1 (GPD1) gene and the effect of HTGTI on the protein structure of GPD1. Methods: Retrospective analysis, using the general data, symptoms, signs, and auxiliary examinations, was performed on patients with HTGTI, which were confirmed by genetic testing in our hospital and reported cases online. The clinical data were analyzed using statistical and bioinformatic approaches. Results: A total of 31 genetically confirmed HTGTI patients were collected from our hospital and cases reported in the literature. The clinical manifestations showed the median age of onset was 6.0 (1.9, 12.0) months. All the patients had normal psychiatric status, but 22.6% of them presented growth retardation and short stature, 93.5% had hepatomegaly, and 16.1% had splenomegaly. Just a few children were reported with jaundice, cholestasis, and obesity (3.2-6.5%). The laboratory investigations showed that 96.8% of them had hypertriglyceridemia (HTG) with a median level of 3.1 (2.1, 5.5) mmol/L, but only 30.0% had returned to normal during follow-up. In addition, 93.5% of patients had elevated alanine aminotransferase (ALT) with an average level of 92.1 ± 43.5 U/L, while 38.7% had hypercholesterolemia. Upon abdominal imaging, all patients presented fatty liver and liver steatosis, with 66.7% of patients showing hepatic fibrosis. Statistical differences in triglyceride (TG) level were observed in the ≤6 months group compared with the older groups and in the 13 months to 6 years group with >6 years group (H = 22.02, P < 0.05). The restricted cubic spline model showed that severe HTG decreased in the early stage of infants to the normal level; however, it rebounded again to a mild or moderate level after the following days. The genetic test revealed that the main variant types of the GPD1 gene were missense variants (51.6%), followed by splicing variants (35.5%) and nonsense variants (12.9%). Of patients, 87.1% had homozygous variants, with the most frequent loci being c.361-1G > C and c.895G > A. Conclusion: The common manifestations of HTGTI were HTG, hepatomegaly, elevated liver transaminases, and hepatic steatosis in early infancy. However, the recurrence of aberrant HTG may pose long-term detrimental effects on HTGTI patients.

All-In-One Biomimetic Nanoplatform Based on Hollow Polydopamine Nanoparticles for Synergistically Enhanced Radiotherapy of Colon Cancer.

Even though radiotherapy is the most important therapeutic strategy for colon cancer treatment, there is an enormous demand to improve radiosensitivity in solid tumor destruction. For this purpose, a biomimetic nanoplatform based on hollow polydopamine nanoparticles (HP) with homologous targeting and pH-responsive drug release properties is designed. In this work, HP is constructed by using a chelation competition-induced polymerization strategy and then modified with the cancer cell membrane. Hollow polydopamine integrated with Pt nanoparticles (Pt@HP) has a catalase-like activity, which can be used to trigger endogenous H2 O2 into O2 , relieving hypoxia of the tumor microenvironment (TME). With mesoporous shells and large cavities, Pt@HP shows efficient apoptin100-109 (AP) and verteporfin (VP) loading to form AVPt@HP@M. Under X-ray irradiation, AVPt@HP@M exerts a radiosensitization effect via multiple strategies, including relieving hypoxia (Pt NPs), enhancing tumor apoptosis (AP), and X-ray-induced photodynamic therapy (X-PDT) (VP). Further metabonomics analysis shows that the specific mechanism of the AVPt@HP@M is through influencing purine metabolism. Without appreciable systemic toxicity, this nanoplatform highlights a new strategy for effective radiosensitization and provides a reference for treating malignant tumors.

Activation and Immune Regulation Mechanisms of PYHIN Family During Microbial Infection.

The innate immune system defenses against pathogen infections via patten-recognition receptors (PRRs). PRRs initiate immune responses by recognizing pathogen-associated molecular patterns (PAMPs), including peptidoglycan, lipopolysaccharide, and nucleic acids. Several nucleic acid sensors or families have been identified, such as RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), cyclic GMP-AMP synthase (cGAS), and PYHIN family receptors. In recent years, the PYHIN family cytosolic DNA receptors have increased attention because of their important roles in initiating innate immune responses. The family members in humans include Absent in melanoma 2 (AIM2), IFN-γ inducible protein 16 (IFI16), interferon-inducible protein X (IFIX), and myeloid cell nuclear differentiation antigen (MNDA). The PYHIN family members are also identified in mice, including AIM2, p202, p203, p204, and p205. Herein, we summarize recent advances in understanding the activation and immune regulation mechanisms of the PYHIN family during microbial infection. Furthermore, structural characterizations of AIM2, IFI16, p202, and p204 provide more accurate insights into the signaling mechanisms of PYHIN family receptors. Overall, the molecular details will facilitate the development of reagents to defense against viral infections.

A partially disordered region connects gene repression and activation functions of EZH2.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2-EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.

A Dimeric Structural Scaffold for PRC2-PCL Targeting to CpG Island Chromatin.

Diverse accessory subunits are involved in the recruitment of polycomb repressive complex 2 (PRC2) to CpG island (CGI) chromatin. Here we report the crystal structure of a SUZ12-RBBP4 complex bound to fragments of the accessory subunits PHF19 and JARID2. Unexpectedly, this complex adopts a dimeric structural architecture, accounting for PRC2 self-association that has long been implicated. The intrinsic PRC2 dimer is formed via domain swapping involving RBBP4 and the unique C2 domain of SUZ12. MTF2 and PHF19 associate with PRC2 at around the dimer interface and stabilize the dimer. Conversely, AEBP2 binding results in a drastic movement of the C2 domain, disrupting the intrinsic PRC2 dimer. PRC2 dimerization enhances CGI DNA binding by PCLs in pairs in vitro, reminiscent of the widespread phenomenon of transcription factor dimerization in active transcription. Loss of PRC2 dimerization impairs histone H3K27 trimethylation (H3K27me3) on chromatin at developmental gene loci in mouse embryonic stem cells.

Unique Structural Platforms of Suz12 Dictate Distinct Classes of PRC2 for Chromatin Binding.

Developmentally regulated accessory subunits dictate PRC2 function. Here, we report the crystal structures of a 120 kDa heterotetrameric complex consisting of Suz12, Rbbp4, Jarid2, and Aebp2 fragments that is minimally active in nucleosome binding and of an inactive binary complex of Suz12 and Rbbp4. Suz12 contains two unique structural platforms that define distinct classes of PRC2 holo complexes for chromatin binding. Aebp2 and Phf19 compete for binding of a non-canonical C2 domain of Suz12; Jarid2 and EPOP occupy an overlapped Suz12 surface required for chromatin association of PRC2. Suz12 and Aebp2 progressively block histone H3K4 binding to Rbbp4, suggesting that Rbbp4 may not be directly involved in PRC2 inhibition by the active H3K4me3 histone mark. Nucleosome binding enabled by Jarid2 and Aebp2 is in part accounted for by the structures, which also reveal that disruption of the Jarid2-Suz12 interaction may underlie the disease mechanism of an oncogenic chromosomal translocation of Suz12.

Response to Comment on "Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2".

Zhang et al suggested that in the crystal structure of a polycomb repressive complex 2 from Chaetomium thermophilum (ctPRC2), a flexible linker region, but not the H3K27M cancer mutant peptide, better fits the electron density. Based on our new data, we agree with this alternative interpretation and provide the crystal structure of ctPRC2 bound to a bona fide H3K27M sequence.

Structural analysis of an active fungal PRC2.

Crystal structure determination of an active polycomb repressive complex 2 (PRC2) from a thermophilic fungus, Chaetomium thermophilum, revealed some long-sought structural mechanisms for assembly, catalysis, and regulation of this important enzyme complex, responsible for trimethylation of histone H3K27 (H3K27me3) and silencing of developmentally regulated genes. In light of the crystal structures of the fungal PRC2 captured in the basal and H3K27me3-stimulated states as well as the structural analysis published previously, (1) we examined surface conservation and electrostatic potential distribution to provide additional insights into functional similarity and divergence between the fungal and human PRC2 and for PRC2 binding by nucleic acids. Structure comparison indicated a conformational change of the catalytic SET domain within PRC2 during transition from the inactive to active state. This conserved structural mechanism is also used by another histone methyltransferase family associated with gene activation for enzyme regulation and may underlie the allosteric stimulation of PRC2 as well.

Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27 trimethylation (H3K27me3), a hallmark of gene silencing. Here we report the crystal structures of an active PRC2 complex of 170 kilodaltons from the yeast Chaetomium thermophilum in both basal and stimulated states, which contain Ezh2, Eed, and the VEFS domain of Suz12 and are bound to a cancer-associated inhibiting H3K27M peptide and a S-adenosyl-l-homocysteine cofactor. The stimulated complex also contains an additional stimulating H3K27me3 peptide. Eed is engulfed by a belt-like structure of Ezh2, and Suz12(VEFS) contacts both of these two subunits to confer an unusual split active SET domain for catalysis. Comparison of PRC2 in the basal and stimulated states reveals a mobile Ezh2 motif that responds to stimulation to allosterically regulate the active site.

Astemizole arrests the proliferation of cancer cells by disrupting the EZH2-EED interaction of polycomb repressive complex 2.

Polycomb Repressive Complex 2 (PRC2) modulates the chromatin structure and transcriptional repression by trimethylation lysine 27 of histone H3 (H3K27me3), a process that necessitates the protein-protein interaction (PPI) between the catalytic subunit EZH2 and EED. Deregulated PRC2 is intimately involved in tumorigenesis and progression, making it an invaluable target for epigenetic cancer therapy. However, until now, there have been no reported small molecule compounds targeting the EZH2-EED interactions. In the present study, we identified astemizole, an FDA-approved drug, as a small molecule inhibitor of the EZH2-EED interaction of PRC2. The disruption of the EZH2-EED interaction by astemizole destabilizes the PRC2 complex and inhibits its methyltransferase activity in cancer cells. Multiple lines of evidence have demonstrated that astemizole arrests the proliferation of PRC2-driven lymphomas primarily by disabling the PRC2 complex. Our findings demonstrate the chemical tractability of the difficult PPI target by a small molecule compound, highlighting the therapeutic promise for PRC2-driven human cancers via targeted destruction of the EZH2-EED complex.

Structural and biochemical characterization reveals LysGH15 as an unprecedented "EF-hand-like" calcium-binding phage lysin.

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

Mechanism of the Rpn13-induced activation of Uch37.

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage.

Asparaginyl endopeptidase (AEP) is an endo/lysosomal cysteine endopeptidase with a preference for an asparagine residue at the P1 site and plays an important role in the maturation of toll-like receptors 3/7/9. AEP is known to undergo autoproteolytic maturation at acidic pH for catalytic activation. Here, we describe crystal structures of the AEP proenzyme and the mature forms of AEP. Structural comparisons between AEP and caspases revealed similarities in the composition of key residues and in the catalytic mechanism. Mutagenesis studies identified N44, R46, H150, E189, C191, S217/S218 and D233 as residues that are essential for the cleavage of the peptide substrate. During maturation, autoproteolytic cleavage of AEP's cap domain opens up access to the active site on the core domain. Unexpectedly, an intermediate autoproteolytic maturation stage was discovered at approximately pH 4.5 in which the partially activated AEP could be reversed back to its proenzyme form. This unique feature was confirmed by the crystal structure of AEPpH4.5 (AEP was matured at pH 4.5 and crystallized at pH 8.5), in which the broken peptide bonds were religated and the structure was transformed back to its proenzyme form. Additionally, the AEP inhibitor cystatin C could be digested by the fully activated AEP, but could not be digested by activated cathepsins. Thus, we demonstrate for the first time that cystatins may regulate the activity of AEP through substrate competition for the active site.

Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation.

Negative-stranded RNA viruses cover their genome with nucleoprotein (N) to protect it from the human innate immune system. Abrogation of the function of N offers a unique opportunity to combat the spread of the viruses. Here, we describe a unique fold of N from Leanyer virus (LEAV, Orthobunyavirus genus, Bunyaviridae family) in complex with single-stranded RNA refined to 2.78 Å resolution as well as a 2.68 Å resolution structure of LEAV N-ssDNA complex. LEAV N is made up of an N- and a C-terminal lobe, with the RNA binding site located at the junction of these lobes. The LEAV N tetramer binds a 44-nucleotide-long single-stranded RNA chain. Hence, oligomerization of N is essential for encapsidation of the entire genome and is accomplished by using extensions at the N and C terminus. Molecular details of the oligomerization of N are illustrated in the structure where a circular ring-like tertiary assembly of a tetramer of LEAV N is observed tethering the RNA in a positively charged cavity running along the inner edge. Hydrogen bonds between N and the C2 hydroxyl group of ribose sugar explain the specificity of LEAV N for RNA over DNA. In addition, base-specific hydrogen bonds suggest that some regions of RNA bind N more tightly than others. Hinge movements around F20 and V125 assist in the reversal of capsidation during transcription and replication of the virus. Electron microscopic images of the ribonucleoprotein complexes of LEAV N reveal a filamentous assembly similar to those found in phleboviruses.

Structure of severe fever with thrombocytopenia syndrome virus nucleocapsid protein in complex with suramin reveals therapeutic potential.

Severe fever with thrombocytopenia syndrome is an emerging infectious disease caused by a novel bunyavirus (SFTSV). Lack of vaccines and inadequate therapeutic treatments have made the spread of the virus a global concern. Viral nucleocapsid protein (N) is essential for its transcription and replication. Here, we present the crystal structures of N from SFTSV and its homologs from Buenaventura (BUE) and Granada (GRA) viruses. The structures reveal that phleboviral N folds into a compact core domain and an extended N-terminal arm that mediates oligomerization, such as tetramer, pentamer, and hexamer of N assemblies. Structural superimposition indicates that phleboviral N adopts a conserved architecture and uses a similar RNA encapsidation strategy as that of RVFV-N. The RNA binding cavity runs along the inner edge of the ring-like assembly. A triple mutant of SFTSV-N, R64D/K67D/K74D, almost lost its ability to bind RNA in vitro, is deficient in its ability to transcribe and replicate. Structural studies of the mutant reveal that both alterations in quaternary assembly and the charge distribution contribute to the loss of RNA binding. In the screening of inhibitors Suramin was identified to bind phleboviral N specifically. The complex crystal structure of SFTSV-N with Suramin was refined to a 2.30-Å resolution. Suramin was found sitting in the putative RNA binding cavity of SFTSV-N. The inhibitory effect of Suramin on SFTSV replication was confirmed in Vero cells. Therefore, a common Suramin-based therapeutic approach targeting SFTSV-N and its homologs could be developed for containing phleboviral outbreaks.

S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS.

A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the R(ano)/R(p.i.m.) ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d''/sig(d'') calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the `parameter-space screening method' for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution.